MicroRNA-21 and microRNA-148a affects PTEN, NO and ROS in canine leishmaniasis.

Canine Visceral leishmaniasis (CanL) poses a severe public health threat in several countries. Disease progression depends on the degree of immune response suppression. MicroRNAs (miRs) modulate mRNA translation into proteins and regulate various cellular functions and pathways associated with immune responses. MiR-21 and miR-148a can alter the parasite load and M1 macrophages are the principal cells in dogs' leishmanicidal activity. A previous study found increased miR-21 and miR-148a in splenic leukocytes (SL) of dogs with CanL using microarray analysis and in silico analysis identified PTEN pathway targets. PTEN is involved in the immune regulation of macrophages. We measured PTEN and the production of reactive oxygen species (ROS) and nitric oxide (NO) before and after transfection SLs of dogs with CanL with mimic and inhibition of miR-21 and miR-148a. PTEN levels increased, NO and ROS decreased in SLs from dogs with CanL. Inhibition of miRNA-21 resulted in PTEN increase; in contrast, PTEN decreased after miR-148a inhibition. Nitrite (NO2) levels increased after transfection with miR-21 inhibitor but were decreased with miR-148a inhibitor. The increase in miR-21 promoted a reduction in ROS and NO levels, but miR-148a inhibition increased NO and reduced ROS. These findings suggest that miR-21 and miR-148a can participate in immune response in CanL, affecting PTEN, NO, and ROS levels.

miR-148a regulation interferes in inflammatory cytokine and parasitic load in canine leishmaniasis.

Canine leishmaniasis (CanL) is a severe public health threat. Infected animals mediate transmission of the Leishmania protozoan to humans via the sandfly's bite during a blood meal. CanL progression depends on the degree of suppression of the immune response, possibly associated with microRNAs (miR), which can modulate mRNA translation into proteins and (consequently) regulate cell function. Increased miR-148a in splenic leukocytes (SL) of dogs with CanL was observed in previous studies, and in silico analysis, identified possible pathways involved in immune response regulation that are affected by this miR. Therefore, we evaluated the involvement of miR-148a in the regulation of TNF-α, IL-6, IL-12, IL-1ß, iNOS, MHCII, CD80, CD3, T-bet, and GATA-3 transcription factors and their relationship with parasite load in SL of dogs with CanL. Splenic leukocytes obtained from healthy and diseased dogs were transfected with miR-148a mimic and inhibitor oligonucleotides. After 48 hours, expression levels of MHCII, CD80, iNOS, CD3, T-bet, and GATA-3 were evaluated by flow cytometry, and concentrations of TNF-α, IL-12, IL-6, and IL-1ß were measured in culture supernatants by capture enzyme-linked immunosorbent assays. Transfection of SL with miR-148a mimics decreased iNOS levels in cells and TNF-α, IL-6, and IL-12 in the supernatants of cultured SL from CanL dogs. Interestingly, transfection with miR-148a inhibitor decreased parasite load in SL cells. These results suggest a direct or not regulatory role of this miR in the immune response to Leishmania infantum infection. We conclude that miR-148a can modulate immune responses by regulating inflammatory cytokines during CanL. Our results contribute to understanding the complex host/parasite interaction in CanL and could assist the development of treatments.

miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis.

Visceral leishmaniasis in humans is a chronic and fatal disease if left untreated. Canine leishmaniasis (CanL) is a severe public health problem because infected animals are powerful transmitters of the parasite to humans via phlebotomine vectors. Therefore, dogs are an essential target for control measures. Progression of canine infection is accompanied by failure of cellular immunity with reduction of circulating lymphocytes and increased cytokines that suppress macrophage function. Studies showed that the regulation of the effector function of macrophages and T cells appears to depend on miRNAs; miRNA-21 (miR-21) shows increased expression in splenic leukocytes of dogs with CanL and targets genes related to the immune response. Mimics and inhibitors of miR-21 were used in vitro to transfect splenic leukocytes from dogs with CanL. After transfection, expression levels of the proteins FAS, FASL, CD69, CCR7, TNF-α, IL-17, IFN-γ, and IL-10 were measured. FAS, FASL, CD69, and CCR7 expression levels decreased in splenic leukocytes from dogs with CanL. The miR-21 mimic decreased CD69 expression in splenic leukocytes from CanL and healthy groups. The miR-21 inhibitor decreased IL-10 levels in culture supernatants from splenic leukocytes in the CanL group. These findings suggest that miR-21 alters the immune response in CanL; therefore, miR-21 could be used as a possible therapeutic target for CanL.

Evaluation of hematological, biochemical and oxidative stress profile in calves under propofol anesthesia.

Propofol is a widely used drug in veterinary medicine to induce anesthesia; as well as the chosen compound for protocols of intravenous anesthesia. The present study aimed to describe the hematological, biochemical and oxidative stress alterations in calves kept under anesthesia by propofol in different dosages. In order to achieve this, eight Holstein calves were induced using propofol in a 5 mg/kg dosage and maintained under continuous propofol infusion for 60 min, having being administered 0.6 mg/kg/h or 0.8 mg/kg/h in crossover design with seven days interval. Blood samples were collected immediately before the anesthesia induction (baseline), and 30 min, 1, 2, 3, 4 and 5 h after the procedure started. Statistically relevant propofol influence was observed both in blood and biochemical parameters, with differences between dosages according to the time of infusion. The drug action over oxidative stress was also observed, causing a raise of the total antioxidant capacity (TAC) with an uric acid increase. Additionally, the increase of triglycerides, induced by the anesthesia maintenance with propofol, caused lipemia in the samples, which was capable of interfering directly in the measurements made by refractometry and spectrophotometry. It was concluded that, in spite of propofol induced alterations in blood and biochemical parameters, such alterations are subtle. In addition to that, the drug presented an antioxidative effect, which reinstates the safety of anesthesia maintenance with propofol in calves.

PD-1 regulates leishmanicidal activity and IL-17 in dogs with leishmaniasis.

Leishmaniasis is an immunosuppressive disease caused by protozoa of the genus Leishmania, for which dogs are the domestic reservoir. The programmed cell death-1 molecule (PD-1) is highly expressed in leukocyte cells of dogs with leishmaniasis, and it promotes T lymphocyte exhaustion and suppression of cytokine secretion. Because PD-1 has a suppressive function regarding cell immunity, we evaluated the effect of PD-1 blocking antibodies on NO, ROS and interleukin 17 (IL-17) production and on parasite load in spleen leukocyte cultures from dogs with leishmaniasis. In vitro, PD-1 blocking promoted increased levels of intracellular NO and NO2 and reduced the levels of IL-17 in the culture supernatant, in addition to reducing the parasite load, but it did not change ROS levels. We conclude that PD-1 participates in the regulation of the immune response and that the blocking antibody is effective in restoring host microbicidal activity. This can be investigated in an immunotherapeutic study in the future.

PD-1 and PD-L1 regulate cellular immunity in canine visceral leishmaniasis.

PD-1 is a negative costimulator of chronic infectious diseases In this study, we investigated the expression of PD-1 and its ligands in the spleen of dogs with visceral leishmaniasis and lymphoproliferative response to soluble antigen, in lymph node cells in the presence or absence of antibodies blocking PD-1 and its ligands. Our results showed expression of PD-1 and its ligands is higher after L. infantum infection and in the spleen of infected dogs, PD-1 blockage was able to restore the antigen-dependent lymphoproliferative response and regulated production of the cytokines IL-4 and IL-10 and NO production. We concluded that L. infantum infection modulates PD-1 and its ligands expression in canine VL and that blockage of PD-1 restores the immune response. Thus, blockage of PD-1 is a target for therapeutic drug development.

Cellular apoptosis and nitric oxide production in PBMC and spleen from dogs with visceral leishmaniasis.

Nitric oxide (NO) is involved in the death of the Leishmania parasite and regulation of apoptosis. We quantified the frequency of cells producing NO and its levels in the peripheral blood mononuclear cells (PBMC), leukocytes from spleen in Visceral Leishmaniasis (VL) symptomatic dogs and correlated NO levels with apoptosis and parasite load in the spleen. The percentage of NO+ cells and CD14+/NO+ was higher in PBMC and spleen cells in infected dogs than in controls. The levels of NO+ and CD14+/NO+ cells was higher in PBMC, but lower spleen of dogs infected than compared to control. Late apoptosis rates increased in PBMC and spleen of infected dogs compared to controls, and the NO levels and apoptosis not showed correlation. There was a positive correlation between the percentage of cells producing NO in the spleen and parasite load. The NO participates in the immune response in the canine VL, but it is not apoptosis inducer.

Stress hormones concentrations in the normal microenvironment predict risk for chemically induced cancer in rats.

Evidence show that stress hormones can influence cancer progression, but its role in carcinogenesis is poorly understood. In this study, we used a new method based on oral carcinogenesis model in rats to test the hypothesis that physiological levels of stress hormones in the normal tissue microenvironment would have significant predictive value for chemically induced cancer occurrence. Male Wistar rats were submitted to a tongue biopsy for measuring not-stress induced levels of norepinephrine, corticosterone, adrenocorticotropic hormone (ACTH) and brain-derived neurotrophic factor (BDNF) in the tissue before carcinogenic induction. Rats were treated with the 4-nitroquinoline-1-oxide (4NQO) chemical carcinogen for twenty weeks and then euthanized for microscopic evaluation of the tongue lesions. Increased pre-carcinogen norepinephrine concentrations and reduced basal corticosterone levels in the normal tissue microenvironment were predictive for oral squamous cell carcinoma (OSCC) occurrence. Likewise, increased pre-carcinogen norepinephrine levels in the normal microenvironment were associated a lower expression of pCDKN2a-p16 in OSCCs. Post-carcinogen levels of corticosterone and BDNF in oral leukoplakia tissues (precursor lesion of OSCC) and post-carcinogen corticosterone concentrations in OSCCs were higher than basal levels in the normal mucosa. Increased norepinephrine concentrations in OSCCs were associated to a greater tumor volume and thickness. Furthermore, higher levels of norepinephrine, ACTH and BDNF in OSCCs were associated to a lesser intensity of the lymphoplasmocytic infiltrate. This study shows that pre-carcinogen stress hormones levels in the normal microenvironment may be predictive for chemically induced cancer in rats. Moreover, chemical carcinogenesis can promote stressor-like effects with hormonal changes in the tissue microenvironment, which may be associated to tumor progression.

Bispectral index assessment in calves subjected to the continuous infusion of propofol combined with fentanyl administration / Indice biespectral em bezerros submetidos à infusão contínua de propofol associado ao fentanil

ABSTRACT: The aim of this study was to evaluate the bispectral index (BIS) effects in calves through continuous infusion of propofol with or without fentanyl. Eight Holstein male calves (ages from six to twelve months old) with an average weight of 123±18kg were used. All animals participated in both groups, always keeping a minimum interval of one week between the anesthetic procedures; the calves were randomly distributed between groups. Anesthesia was induced with an intravenous (IV) dose of propofol of 5mg kg-1 in control group (GP) or with propofol (4mg kg-1) associated with IV fentanyl 0.001mg kg-1(GF). All the calves were positioned in right lateral recumbency and were allowed to spontaneously breathe room air. Subsequently, the anesthesia was maintained by continuous infusion of propofol at the rate of 0.6mg kg-1 min-1 IV in GP, and associated with the infusion of fentanyl 0.001mg kg-1 hour-1 in GF. Measurements of BIS, signal quality index (SQI) and electromyography (EMG) were evaluated before anesthesia induction (TB), and at 15, 30, 45 and 60 minutes after the beginning of continuous drugs infusion (T15, T30, T45 and T60, respectively). The heart rate (HR), respiratory rate (f), end-tidal carbon dioxide tension (ETCO2) and recovery times were evaluated as well. No significant differences were observed between the groups in the BIS variables and the recovery time was longer in GF. Co-administration of propofol and fentanyl infusions, at the doses reported here, did not change the values of BIS in cattle, but delayed the recovery time.

RESUMO: O estudo teve por objetivo avaliar o índice biespectral (BIS) durante a infusão contínua de propofol associado ou não ao fentanil em bezerros. Foram utilizados oito animais machos entre seis e doze meses de idade, holandeses, com massa corporal média de 123±18kg. Todos os animais participaram de ambos os grupos, respeitando-se sempre um intervalo mínimo de uma semana entre uma anestesia e outra, sendo aleatoriamente distribuídos entre os grupos. A anestesia nos bezerros foi induzida com propofol na dose de 5mg kg-1; intravenoso (IV), grupo controle (GP) ou propofol 4mg kg-1 associado ao fentanil 0,001mg kg-1; IV, grupo fentanil (GF) e posicionados em decúbito lateral direito, onde permaneceram respirando espontaneamente ar ambiente. Ato contínuo, a manutenção anestésica foi realizada pela infusão contínua de propofol na taxa de 0,6mg kg-1 min-1; IV GP, associado ou não à infusão de fentanil 0.001mg kg-1 hora-1 GF. A mensuração das variáveis do BIS, índice de qualidade de sinal (IQS) eletromiografia (EMG), frequência cardíaca (FC), frequência respiratória (f) e dióxido de carbono ao final da expiração (ETCO2) foram avaliadas antes da indução anestésica no momento basal (MB), e 15, 30, 45 e 60 minutos após o início da infusão contínua dos fármacos (M15, M30, M45 e M60, respectivamente); o tempo de recuperação também foi avaliado. Não foram observadas diferenças significativas entre os grupos nas variáveis do BIS e o tempo de recuperação foi maior no GF. A co-administração das infusões de propofol e fentanil, nas doses utilizadas nesse estudo, não alterou os valores do BIS em bezerros, porém, prolongou o tempo de recuperação.

The effects of increased heme oxygenase-1 on the lymphoproliferative response in dogs with visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is known to affect the cellular immunity of infected dogs, through impairing lymphoproliferation and microbicidal mechanisms. This study examined heme oxygenase-1 (HO-1) and its metabolites, oxidative stress and IL-10 levels in CVL and investigated correlations between these parameters. Additionally, the effects of HO-1 inhibition on the lymphoproliferative response and cytokine production in lymph node cells (LNCs) from infected dogs were evaluated. Forty-four dogs, 24 controls and 20 dogs with CVL were selected. Plasma and splenic levels of HO-1, haptoglobin, soluble CD163 receptor, ferritin and IL-10 were determined using capture ELISA. The HO-1 levels and relative gene expression in peripheral blood and bone marrow mononuclear cells were also determined. LNCs proliferation was evaluated with an HO-1 activator and with an HO-1 inhibitor, in the presence of the Leishmania infantum soluble antigen (SAgL), using flow cytometry. HO-1, IL-2, IFN-gamma and IL-10 were also determined in these cultures using capture ELISA. Infected dogs presented oxidative stress and increased HO-1 levels and relative gene expression, with correlation between oxidative stress and HO-1. The substances from heme metabolism and IL-10 were also elevated in the plasma and spleens of infected dogs. IL-10 and HO-1 levels were positively correlated with one another. Inhibition of HO-1 increased LNCs proliferation and decreased IL-10 and IL-2 production in the presence of SAgL. The increased HO-1 metabolism observed in CVL is probably associated with oxidative stress and increased IL-10, which could be one of the mechanisms responsible for inhibition of the lymphoproliferative response in sick dogs.

PD-1 function in apoptosis of T lymphocytes in canine visceral leishmaniasis.

Dogs infected with Leishmania infantum have a reduced number of T lymphocytes. PD-1 (Programmed cell death 1) a new member of the B7-CD28 family that is expressed by immune cells, and its binding to PD-L1 (CD274) or PD-L2 (CD273) induces the deactivation or apoptosis of T cells. This study aimed to evaluate the expression of PD-1 and its ligands, as well as blocking in the induction of apoptosis in T lymphocytes, TNF-α, IL-4 and nitric oxide production by leucokocytes from PBMC and spleen and the parasite load in dogs with visceral leishmaniasis (VL). Our results showed that the expression of PD1 and its ligands was increased in CD3(+) T cells and CD21(+) B lymphocytes within the peripheral blood and splenic mononuclear cells of dogs with VL. In peripheral blood monocytes, only PD-1 ligands exhibited increased expression; however, in spleen macrophages, increased expression of both PD-1 and its ligands was observed. Levels of apoptosis in peripheral blood and splenic T lymphocytes were higher in dogs with VL compared to healthy dogs. Blocking monoclonal antibodies to PD-1 and its ligands in the culture of mononuclear cells from the peripheral blood and spleen decreased the amount of CD3(+) T lymphocyte apoptosis. The concentration of nitric oxide, TNF-α and IL-4 increased in the culture supernatants of peripheral blood mononuclear cells treated with a blocking monoclonal antibody against PD-1. The TNF-α concentration increased in the culture supernatants of splenic cells following all treatments with antibodies blocking PD-1 and its ligands; however, the amount of IL-4 increased only in the presence of a PD-1 blocking agent. Treatment with a PD-1 blocking monoclonal antibody in the mononuclear peripheral blood of dogs with VL reduced the parasite burden while increased TNF-α. We conclude that in canine visceral leishmaniasis, PD-1 and its ligands are involved in the induction of T lymphocyte apoptosis and in regulating the production of nitric oxide, TNF-α, and IL-4, as well as the parasitic load.

Bispectral index in calves anesthetized with xylazine, midazolam, ketamine, isoflurane and subjected to continuous rate infusion of lidocaine.

PURPOSE: To assess the bispectral index (BIS) and recovery in calves anesthetized with xylazine, midazolam, ketamine and isoflurane and subjected to CRI of lidocaine. METHODS: Xilazine was administered followed by ketamine and midazolam, orotracheal intubation and maintenance on isoflurane using mechanical ventilation. Lidocaine (2 mg kg(-1) bolus) or saline (0.9%) was administered i.v. followed by a CRI (100 µg kg(-1) minute(-1)) of lidocaine (L) or saline (C). Were recorded BIS, heart rate (HR), mean arterial pressure (MAP) and rectal temperature (RT) before administration of premedication (TB) and 15 minutes after (TX), before administering lidocaine (T0) and 20, 40, 60 and 80 minutes after the start of the CRI . Time do sternal recumbency (SRE) and standing (ST) and plasma lidocaine concentration also evaluated. RESULTS: In both treatments BIS decreased significantly at all times compared to TB. TX was higher than the subsequent times. HR decreased from baseline at all times and decreased from T40 in L compared to C. SRE was higher in L compared to C. CONCLUSIONS: Bispectral index values were consistent with the degree of hypnosis of the animals. Lidocaine did not potentiate isoflurane anesthesia assessed by BIS in unstimulated calves anesthetized with constant E´Iso. Lidocaine increased the time to sternal recumbency.

Bispectral index in calves anesthetized with xylazine, midazolam, ketamine, isoflurane and subjected to continuous rate infusion of lidocaine

PURPOSE: To assess the bispectral index (BIS) and recovery in calves anesthetized with xylazine, midazolam, ketamine and isoflurane and subjected to CRI of lidocaine. METHODS: Xilazine was administered followed by ketamine and midazolam, orotracheal intubation and maintenance on isoflurane using mechanical ventilation. Lidocaine (2 mg kg-1 bolus) or saline (0.9%) was administered IV followed by a CRI (100 µg kg-1 minute-1) of lidocaine (L) or saline (C). Were recorded BIS, heart rate (HR), mean arterial pressure (MAP) and rectal temperature (RT) before administration of premedication (TB) and 15 minutes after (TX), before administering lidocaine (T0) and 20, 40, 60 and 80 minutes after the start of the CRI . Time do sternal recumbency (SRE) and standing (ST) and plasma lidocaine concentration also evaluated. RESULTS: In both treatments BIS decreased significantly at all times compared to TB. TX was higher than the subsequent times. HR decreased from baseline at all times and decreased from T40 in L compared to C. SRE was higher in L compared to C. CONCLUSIONS: Bispectral index values were consistent with the degree of hypnosis of the animals. Lidocaine did not potentiate isoflurane anesthesia assessed by BIS in unstimulated calves anesthetized with constant E´Iso. Lidocaine increased the time to sternal recumbency. .

Effects of a continuous rate infusion of butorphanol in isoflurane-anesthetized horses on cardiorespiratory parameters, recovery quality, gastrointestinal motility and serum cortisol concentrations.

PURPOSE: To assess the cardiorespiratory parametes, recovery, gastrointestinal motility and serum cortisol concentrations in horses anesthetized with isoflurane with or without a continuous rate infusion (CRI) of butorphanol for orchiectomy. METHODS: Twelve adult, intact, male horses weighing 332 ± 55 kg were included in the study. Xilazine was administered as premedication. Anesthesia was induced with ketamine and midazolam and maintained with isoflurane. Butorphanol (0.025 mg kg-1 bolus) or an equivalent volume of saline (0.9%) was given intravenously followed by a CRI of butorphanol (BG) (13 µg kg-1 hour-1) or saline (CG). Cardiorespiratory variables were recorded before (T0) and every 15 minutes for 75 minutes after the start of infusion. Serum cortisol concentration was measured at T0 and 60 minutes, and 30 minutes and 19 hours after the horse stood up. Recovery from anesthesia was evaluated using a scoring system. Gastrointestinal motility was evaluated before anesthesia and during 24 hours after recovery. RESULTS: There were no significant differences between groups in cardiopulmonary variables, or recovery scores or serum cortisol concentrations. A reduction in gastrointestinal motility was recorded for 60 minutes in BG. CONCLUSIONS: Continuous rate infusion of butorphanol in horses anesthetized with isoflurane did not adversely affect the cardiopulmonary variables monitored, or recovery scores. A small but statistically significant reduction in gastrointestinal motility occurred in the butorphanol group.

Effects of a continuous rate infusion of butorphanol in isoflurane-anesthetized horses on cardiorespiratory parameters, recovery quality, gastrointestinal motility and serum cortisol concentrations

PURPOSE: To assess the cardiorespiratory parametes, recovery, gastrointestinal motility and serum cortisol concentrations in horses anesthetized with isoflurane with or without a continuous rate infusion (CRI) of butorphanol for orchiectomy. METHODS: Twelve adult, intact, male horses weighing 332 ± 55 kg were included in the study. Xilazine was administered as premedication. Anesthesia was induced with ketamine and midazolam and maintained with isoflurane. Butorphanol (0.025 mg kg-1 bolus) or an equivalent volume of saline (0.9%) was given intravenously followed by a CRI of butorphanol (BG) (13 µg kg-1 hour-1) or saline (CG). Cardiorespiratory variables were recorded before (T0) and every 15 minutes for 75 minutes after the start of infusion. Serum cortisol concentration was measured at T0 and 60 minutes, and 30 minutes and 19 hours after the horse stood up. Recovery from anesthesia was evaluated using a scoring system. Gastrointestinal motility was evaluated before anesthesia and during 24 hours after recovery. RESULTS: There were no significant differences between groups in cardiopulmonary variables, or recovery scores or serum cortisol concentrations. A reduction in gastrointestinal motility was recorded for 60 minutes in BG. CONCLUSIONS: Continuous rate infusion of butorphanol in horses anesthetized with isoflurane did not adversely affect the cardiopulmonary variables monitored, or recovery scores. A small but statistically significant reduction in gastrointestinal motility occurred in the butorphanol group. .

Alteration of sFAS and sFAS ligand expression during canine visceral leishmaniosis.

Visceral leishmaniosis (VL) is caused by intracellular parasites of the genus Leishmania that affect humans and several animal species. Dogs are one of the main urban reservoirs of Leishmania infantum and play a central role in the transmission cycle to humans via sandflies. CD3+ cells apoptosis is involved in the immune response in VL. Dysregulation of apoptosis has been implicated in various disease states. An important regulator of apoptosis is the FAS-FAS-associated death domain protein (cluster of differentiation 95 - CD95) and FASL-FAS ligand protein (cluster of differentiation 178 - CD178) system involved in the down-regulation of immune reactions and in T cell-mediated cytotoxicity. FAS is a member of the tumor necrosis factor (TNF) receptor super family, which can be expressed in transmembrane or soluble forms. The soluble levels of FAS (sFAS), FASL (sFASL) and active Caspase-3, this last related to apoptotic cascade, were investigated in the spleen of 19 symptomatic dogs presenting moderate VL and 6 healthy dogs, determined by ELISA assay. The splenic parasite load was determined by real-time PCR monitoring of amplification of the intergenic internal transcribed spacer (ITS1) gene of parasite rRNA. sFAS levels were lower (p<0.05). sFASL and active Caspase-3 levels were higher (p<0.05) in dogs with VL compared with controls. Negative correlation was observed between parasite burden and sFASL levels. The increase in sFASL could be related to the mechanism involved in the elimination of the parasite.

Effects of continuous rate infusion of butorphanol in isoflurane-anesthetized calves.

PURPOSE: To assess the hemodynamic changes and bispectral index (BIS) following administration of a continuous rate infusion (CRI) of butorphanol in isoflurane-anesthetized calves. METHODS: Eight calves weighing 110 ± 12 kg were included in the study. Anesthesia was induced with 5% isoflurane in O2 delivered via face mask and maintained with end-tidal concentration of 1.4%. IPPV was set to a peak inspiratory airway pressure of 15 cmH2O and respiratory rate of six breaths minute-1. Forty minutes after the start of anesthetic maintenance, 0.1 mg kg-1butorphanol was administered intravenously, followed by a CRI of 20 µg kg-1 minute-1. Hemodynamic variables and BIS were recorded before butorphanol administration (T0), and at 10, 20, 40 and 80 minutes following the CRI. Anesthesia was discontinued after the last recording and the calves were allowed to recover. The time to sternal recumbency (SRE) and standing (ST) were evaluated. RESULTS: There were no significant differences between the moments in all hemodynamic variables and BIS. The time to SRE and ST was 9 ± 5 and 14 ± 7 minutes, respectively. CONCLUSION: The continuous rate infusion did not produce clinically relevant changes in hemodynamic or bispectral index values compared to baseline in mechanically ventilated and unstimulated calves anesthetized at 1.4% isoflurane.

Effects of continuous rate infusion of butorphanol in isoflurane-anesthetized calves

PURPOSE: To assess the hemodynamic changes and bispectral index (BIS) following administration of a continuous rate infusion (CRI) of butorphanol in isoflurane-anesthetized calves. METHODS: Eight calves weighing 110 ± 12 kg were included in the study. Anesthesia was induced with 5% isoflurane in O2 delivered via face mask and maintained with end-tidal concentration of 1.4%. IPPV was set to a peak inspiratory airway pressure of 15 cmH2O and respiratory rate of six breaths minute-1. Forty minutes after the start of anesthetic maintenance, 0.1 mg kg-1butorphanol was administered intravenously, followed by a CRI of 20 µg kg-1 minute-1. Hemodynamic variables and BIS were recorded before butorphanol administration (T0), and at 10, 20, 40 and 80 minutes following the CRI. Anesthesia was discontinued after the last recording and the calves were allowed to recover. The time to sternal recumbency (SRE) and standing (ST) were evaluated. RESULTS: There were no significant differences between the moments in all hemodynamic variables and BIS. The time to SRE and ST was 9 ± 5 and 14 ± 7 minutes, respectively. CONCLUSION: The continuous rate infusion did not produce clinically relevant changes in hemodynamic or bispectral index values compared to baseline in mechanically ventilated and unstimulated calves anesthetized at 1.4% isoflurane. .

CD95 (FAS) and CD178 (FASL) induce the apoptosis of CD4+ and CD8+ cells isolated from the peripheral blood and spleen of dogs naturally infected with Leishmania spp.

Infected dogs are urban reservoirs of Leishmania chagasi, which is a causative agent of visceral leishmaniasis (VL). Dogs exhibit immune suppression during the course of this disease, and lymphocyte apoptosis is involved in this process. To investigate apoptosis and the expression levels of FAS-FAS-associated death domain protein (CD95 or APO-1), FASL-FAS ligand protein (CD178), and TRAIL-TNF-related apoptosis-inducing ligand (CD253) receptors in peripheral blood mononuclear cells and spleen leukocytes from 38 symptomatic dogs with moderate VL and 25 healthy dogs were evaluated by flow cytometry. The apoptosis rate of blood and splenic CD4+ and CD8+ cells was higher in infected dogs than in healthy dogs. The expression levels of FAS and FASL in blood and splenic CD4+ cells were lower in infected dogs than in healthy dogs. FAS expression in CD8+ cells was higher in infected dogs than in healthy dogs; in contrast, FASL expression was lower in infected dogs. The expression of the TRAIL receptor increased only in splenic CD8+ cells from infected dogs. The FAS and FAS-L blocking antibodies confirmed the importance of these receptors in apoptosis. Our results enhance the current understanding of the immune response in dogs infected with L. chagasi, facilitating the future development of therapeutic interventions to reduce lymphocyte depletion.

Apoptosis in T lymphocytes from spleen tissue and peripheral blood of L. (L.) chagasi naturally infected dogs.

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite may cause visceral leishmaniasis, which can also be transmitted to humans. Infected symptomatic dogs show disorganization in the white pulp in spleen tissue and a reduction in T lymphocytes in peripheral blood. To investigate whether apoptosis is involved in white pulp disorganization and diminished T cell counts in peripheral blood, apoptotic T cells from the spleen and peripheral blood of dogs naturally infected with L. (L.) chagasi and presenting clinical manifestations were quantified and compared with healthy dogs. Thirteen symptomatic adult dogs infected by L. (L.) chagasi and six healthy dogs from a nonendemic area (controls) were included in the study. Samples from spleen and peripheral blood were used to quantify apoptosis in CD3 lymphocytes by flow cytometry using Anexin V and Multicaspase kits; the results were compared using the Mann Whitney test. The percentage of total T cells was lower in Leishmania infected dogs compared to healthy controls (P<0.05). Apoptosis levels in T cells from PBMC and spleen were higher in infected dogs than in controls (P<0.05). The least squares method test was used to determine the effect between the degree of structural organization of spleen white pulp and the percentage of apoptosis in the spleen. A significant effect on the level of white pulp morphological disorganization and percentage of apoptosis in spleen T cells was observed (F=20.45; P=0.0014). These data suggest that apoptosis is an important for the immunopathogenesis of canine visceral leishmaniasis.